Replication of HAV is being studied in order to find ways to improve the in vitro replication of the virus. As the virus presently grows slowly and to relatively low titer, vaccine production is inefficient. Several different constructs using the infectious cDNA of HAV as the backbone have been made. These include placing the highly active encephalomyocarditis virus (ECMV) internal ribosomal entry site (IRES) into the 5' non-coding region of the HAV genome. This resulted in a viable virus but without enhanced growth characteristics. We have inserted a multiple cloning site and the cleavage site for 3Cpro just prior to the viral polyprotein initiation site into the infectious cDNA of HAV. As a demonstration. we have now inserted the cDNA coding for an octapeptide, "FLAG peptide" and shown that this construct was both infectious and using a monoclonal antibody against Flag, specific expression was demonstrated. Further modifications in the constructs are being made in order to express larger polypeptides with the aim of being able to express immunuenhancers such as cytokines as part with the virus thus improving immunogenicity. As HAV is a positive stranded RNA virus with no potential for integration and as it does not produce a cytopathic effect of chronic infections, the virus has some ideal characteristics for gene therapy.As part of the search for the cellular receptor for HAV, we investigated the replication of HAV in non-primate cells. It has long been believed that HAV would replicate only in cells of primate origin but it was not know if this was due to lack of the receptor or due to some internal cellular function required for HAV replication. While most cells studied would not support the growth of HAV either by infection or transfection of genomic RNA, we identified 3 cells of swine, guinea pig and dolphin that were infectable with HAV. This result may have utility in live attentuated vaccine development.